Our objective is to investigate the molecular events involved in fibronectin-mediated cell adhesion. Antibinding-site monoclonal antibodies will be prepared which inhibit each of the five known binding sites of fibronectin. We have currently isolated one monoclonal antibody which inhibits the cell adhesive activity of fibronectin by blocking the cell-surface binding site of fibronectin. Using antibinding-site monoclonal antibodies, we will determine whether the five binding sites of fibronectin act in a coordinate fashion. Antibinding-site monoclonal antibodies also will be used to examine the role of each binding site of fibronectin in the process of fibronectin-mediated cell adhesion. Monoclonal antibodies will be developed for the determination of insoluble cellular fibronectin. Such monoclonal reagents should be valuable in the clinical differentiation of malignant from benign tumors. Evidence has been gained which indicates that the glycosaminoglycan binding site of fibronectin plays a role in fibronectin-mediated cell adhesion. We will examine the effects of several cell surface-associated proteoglycans on cell adhesion. The structural features of proteoglycans required for effects on cell adhesion will be determined with monoclonal antibodies and by the isolation of biologically-active fragments of proteoglycans. With the aid of antibinding-site monoclonal antibodies, we will select for mutant cells which synthesize fibronectin molecules with genetically altered binding sites. We will determine whether a mutation affecting one site also influences the biological activity of other sites. The biological significance of fibronectin possessing five different binding sites is currently unknown. Our studies should identify the existence of any coordinate interactions between different fibronectin binding sites. (A)